Aging affects the number and morphological heterogeneity of rat phrenic motor neurons and phrenic motor axons

Abstract

Diaphragm muscle (DIAm) motor units comprise a phrenic motor neuron (PhMN), the phrenic nerve and the muscle fibers innervated, with the size of PhMNs and axons characteristic of motor unit type. Smaller PhMNs and their axons comprise slow (type S) and fatigue-resistant (type FR) DIAm motor units, while larger PhMNs and their axons comprise more fatigable (type FF) motor units. With aging, we have shown a loss of larger PhMNs, consistent with selective atrophy of type IIx/IIb DIAm fibers and reduced maximum DIAm force. In the present study, we hypothesized that with aging there is a loss of larger myelinated phrenic α motor axons. Female and male young (6 months) and old (24 months) Fischer 344 rats were studied. PhMNs were retrogradely labeled by intrapleural injection of 488-conjugated CTB. The phrenic nerves were excised ~1 cm from the DIAm insertion and mounted in resin, and phrenic α motor axons were delineated based on size (i.e., >4 μm diameters). In older rats, the number of larger PhMNs and larger phrenic α motor axons were reduced. There were no differences in non-α axons. In addition, there was evidence of demyelination of larger phrenic α motor axons in older rats. Together, these findings are consistent with the selective age-related vulnerability of larger PhMNs and denervation of type FF motor units, which may underlie DIAm sarcopenia.

Keywords: aging; motor neurons; neurodegeneration; respiratory system.

FIGURE 1

Assessment of phrenic motor neurons (PhMNs) and phrenic α motor axons in young and old rats. (a, b) Example fluorescent photomicrographs of PhMNs of young (6 months) and old (24 months) F344 rats, respectively. (c, d) Example survey EM photomicrographs of phrenic α motor axons of young (6 months) and old (24 months) F344 rats, respectively. (e) Scatterplots show Z‐scores (median ± inter‐quartile range) for PhMN surface area SA (green circles) and phrenic α motor axon cross‐sectional area (CSA) (black circles) from each 6‐month‐old rat sample. Kolmogorov–Smirnov comparisons with each z‐distribution of each young rat showing no differences in size heterogeneity between PhMNs and phrenic α motor axons. Each mean comprises high‐magnification PhMN SA estimates from a minimum of 30 (every 5th) PhMNs/rat/age and high‐magnification phrenic α motor axon CSA estimates from each counted axon.

FIGURE 2

Loss of phrenic motor neurons (PhMNs) and phrenic α motor axons in old rats. (a) Scatterplot shows reduced number of PhMNs (green bars) and phrenic α motor axons (gray bars) in old (blue symbols) compared to young (orange symbols) F344 rats. Within each age group, there was good agreement between PhMN and phrenic α motor axon numbers. Two‐way ANOVA with Bonferroni post hoc test, p < 0.05, n indicated by symbol. (b) XY plot shows the number of PhMNs plotted against the number of phrenic α motor axons within each rat. Linear regression shows excellent agreement between the two estimates (slope = 0.90, p = 0.0002, r ² = 0.84).

FIGURE 3

Reduced phrenic motor neuron (PhMN) somal surface area and phrenic α motor axon cross‐sectional area in old rats. (a, b) An example high‐magnification EM photomicrographs of phrenic α motor axons of young (6 months) and old (24 months) F344 rats, respectively. (c) Scatterplot shows reduced of phrenic α motor axon cross‐sectional area in old (dark gray circles) compared to young (open circles) F344 rats. (d, e) Example fluorescent photomicrographs of PhMNs of young (6 months) and old (24 months) F344 rats, respectively. (f) Scatterplot shows reduced PhMN surface area (SA) in old (dark gray squares) compared to young (open squares) F344 rats. Student's unpaired t‐tests, p < 0.05, n indicated by symbol. Each mean comprises high‐magnification PhMN SA estimates from a minimum of 30 (every 5th) PhMNs/rat/age and high‐magnification phrenic α motor axon CSA estimates from each counted axon.

FIGURE 4

Larger phrenic α motor axons are disproportionately lost in old rats. (a) Reduced % of total phrenic α motor axons that are of larger size in old (dark gray bars) compared to young (light gray bars) F344 rats. Kolmogorov–Smirnov test, p < 0.0001. (b) Scatterplot shows reduced number of phrenic α motor axons in the medial and larger axon size tertile in old (24 months, squares), compared to young (6 months, circles) F344 rats. Two‐way ANOVA with Bonferroni post hoc test, p < 0.05, n indicated by symbol.

FIGURE 5

Reduced myelination in phrenic α motor axons in old rats. (a) An example high‐magnification EM photomicrograph of a phrenic α motor axon adjacent to other non‐α motor myelinated axons. (b) Scatterplot shows reduced mean myelination (μm²) of phrenic α motor axons in old (24 months, squares), compared to young (6 months, circles) F344 rats. Two‐way ANOVA with Bonferroni post hoc test, p < 0.05, n indicated by symbol. Each mean comprises high‐magnification myelin cross‐sectional area estimates from a minimum of 26 axons/rat/age.